Enzymes from armillaria mellea

ABSTRACT

An enzymic substance, AM protease, having fibrinogenolytic, fibrinolytic and anticoagulant activity. Processes for preparing AM protease. Pharmaceutical compositions containing AM protease.

United States Patent [:91

Broadbent et al. [4 1 Jan. 30, 1973 [54] ENZYMES FROM ARMILLARI A 195/66B MELLEA [51] Int. Cl. ..Cl2d 13/10, A6lk 19/00 [75] Inventors: DouglasBro-bent, Alderley Edge [58] Field of Search ..l95/62, 65,66 R, 66 BCheshire; Ralph William Turner, Cheadle, Cheshire; Peter Leslie [56]References C'ted Walton, Knutsford, Cheshire, all of UNITED STATESPATENTS England Ass g e Imperial i a Industries 3,256,l 57 6/l966 Truantet al. ..i95/66 B Lmmed London England Primary ExaminerLionel M. Shapiro[22] Filed: Aug. 31, 1970 Attorney-Cushman, Darby & Cushman 21 A LN68501 1 PP 0 i 57 ABSTRACT [30] Foreign Application priority Data Anenzymic substance, AM protease, having I fibrinogenolytic, fibrinolyticand anticoagulant activi- Sept. 26, 1969 Great Br ta n ..47,554/69processes for preparing AM proteasa pharmacew May 7, I970 1 Great Bntam..22,l29/70 flea] Compositions containing AM prom-33a [52] US. Cl..l95/62, 195/66 R, 424/94, Claims, No Drawings 1 ENZYMES FROMARMILLARIA MELLEA We have found that the mature fruiting body, or cap,

of the fungus Armillaria mellea is a source of a useful enzymicsubstance which is the subject of this applica- 1 tion. Armillariamellea grows parasitically or saprophytically on trees, and it occursthroughout the United Kingdom and in other countries of the world. Abrief description of the fungus is as follows:

Pileus 2-6 inches across, at first rounded, then fiattened, andeventually depressed in the center. Color varies from yellowish to deepbrown. Stem 3 to 6 inches long and up to one-half inch thick. Ringwhitish. Gills whitishto flesh colored, adnate or slightly decurrent.Spores colorless under the microscope, 8-9 X 5-6;4.. The fruiting bodiesare best harvested, so as to obtain the said enzymic substance, fromabout August to the first frosts. After harvesting, the fruiting bodiesshould be stored at about -C. or lower until the isolation proceduredescribed below is carried out.

We have found that the said enzymic substance is not obtainable from allspecimens of Armillaria mellea.

Thus, we obtained the said enzymic substance from a little over onethird (approximately 50 out of a totalof approximately 140) of thesamples'of the fungus which we collected in the United Kingdom during1969. The reasons why some samples did not afford the said enzymicsubstance are unknown to us. Samples of Armil- [aria mellea which areavailable to the public and which afford the said enzymic substance are:I

1. the specimen identified as FPRL 6H by (and available to the publicfrom) the Ministry of Technology, Forest Products Research Laboratories,Princes Risborough, Aylesbury, Buckinghamshire, England (our reference:ACC 3659); and

2. the specimen identified .simply as Armillaria mellea" by (andavailable to the public from) the Centraalbureau voor Schimmelcultures,Baarn, Netherlands (our reference: ACC 3253).

' According to the invention there is provided the said enzymicsubstance which is a peptidyl-peptidehydrolase (syn.peptidePpeptido-hydrolase), and which we shall refer to as AM proteasein this specification. AM protease has the following characteristicproperties:

1. It catalyses the hydrolytic degradation of fibrinogen and fibrin(i.e. it exhibits fibrino genolytic and fibrinolytic activityrespectively). The activity of AM protease in vitro has been determinedby the following known tests:

a. Incubation of AM protease with purified fibrinogen and the additionof thrombin to aliquots after intervals. The time required for the lossof clotting function is an index of fibrinogenolytic activity.

b. lncubation of pre-formed fibrin clots with AM protease, and themeasurement of the rate of clot disappearance. This measurement may befacilitated by preparing clots from I -labelled fibrinogen and measuringthe release of radio-activity. This gives an index of fibrinolyticactivity.

c. 'Mixing AM protease, thrombin and fibrinogen together and observingthe life of the clot formed. This gives a measure of thefibrinogenolytic and fibrinolytic activity of AM protease.

In vivo activity of AM protease in laboratory animals (rabbits) isdetermined by measuring the effect thereof on the fibrinogen levels inthe animals and on the level of protease in the plasma. The former canbe determined by measuring the prolongation of the whole blood or plasmaclotting time, compared with the control or pre-treatment clotting time,on addition of a standard quantity of thrombin. The latter can bedetermined by a method similar to that listed in l (b).

2. AM protease behaves as a single component on passage through a columnof cross-linked dextran gel (Sephadex G 150; Sephadex is a Trade Mark)which is permeable to proteinsof a molecular weight of up to 400,000.The volume of buffer (eluant) in which AM protease emerges from thecolumn corresponds to that required to elute a protein with a molecularweight of about 30,000. The said eluant is 0.3M NaCl 0.05M pH 7.4tris/HCl buffer, which has the following composition (and the elution iscarried out at about 2C.):

Sodium chloride l7.5g./l. in water Tris (hydroxymethyl)aminomethaneSufficient M hydrochloric acid to give pH 7.4

3. AM protease behaves as a single component on sedimentation at 26 X 10X g in the cell of an analytical ultracentrifuge. The sedimentationcoefficient of AM protease in the 0.3M NaCl 0.05M pH 7.4 tris/HCl buffer[see (2) above] at 20C., i.e. the 8 W, is about 2.35 X 10* sec.-

4. Only one component is evident upon electrophoresis of AMprotease inpolyacrylamide gel, and

this component has a migration corresponding to a 'yglobulin. Thepolyacrylamide gel had an acrylamide to bisacrylamide ratio of :1, theelectrophoresis was run for 1 hour at 2 mA./sq. cm., and the buffer used[pH 4.3, B-alanine-acetic acid (0.3M)] consisted of B- alanine (26.73g./ l in water) which was adjusted to pH 4.3 by the addition of glacialacetic acid.

5. The N terminal amino acid of AM protease is isoleucine.

6. AM protease readily degrades casein at pH 7 producing fragmentssoluble in trichloro-acetic acid; the degree of digestion is much lessthan that produced by trypsin, chymotrypsin or plasmin. It causes littledegradation of serum albumin or 'yglobulin, even on prolongedincubation. It causes extensive degradation of fibrinogen. It has noaction on small molecular weight substrates (e.g.a-N-acetylglycyl-L-lysine methyl ester, a-N-acetyl-L-lysine methylester, a-N-ptoluenesulphonyl-L-arginine methyl ester, a-N-acetyl-L-tyrosine ethyl ester or L-tryptophan ethyl ester) which are commonlyused to characterize trypsin-like or chymotrypsin-like enzymes. It actsupon the B-chain of oxidized insulin causing the following amino endgroups to appear:

6.05g./l. in water v tyrosine, lysine and, to a limited extent, leucine;in this respect also (see above) it differs from trypsin andchymotrypsin.

When obtained by the isolation procedure described below, AM proteaseappears to be essentially homogeneous by the usual biophysical criteria;i.e. sedimentation properties, electrophoretic properties, and behavioron gel filtration and ion exchange columns. In all cases homogeneity isindicated by the appearance of a narrow zone of protein having asymmetrical, almost Gaussian, distribution about a peak. Nevertheless,it is possible that AM protease is a mixture of closely-relatedisofunctional enzymes.

According to a further feature of the invention there is provided aprocess for the manufacture of AM protease, which comprises thefollowing sequence of steps:

i. grinding or otherwise converting mature fruiting bodies of Armillariamellea to a small size, and extracting this material with water so as toobtain an aqueous extract;

ii. precipitating unwanted contaminating proteins from the said aqueousextract by means of at least one water-miscible organic solvent at anappropriate temperature, pH and ionic strength, and removing theprecipitated proteins by conventional means;

iii. precipitating crude AM protease from the solution thereof by addingat least one water-miscible organic solvent to the said solution [i.e.the solution remaining after the removal of the unwanted proteins in iv.partially purifying the crude AM chromatography; and

v. purifying the resulting impure AM protease by means ofa molecularsieve technique. It is to be understood that unless otherwise indicated,all of the above steps are carried out at a temperature of about 2C. Theabove sequence of steps will now be considered in detail.

i. As indicated above, the fruiting bodies of Armillaria mellea aregenerally at the correct stage of maturity during late summer andautumn. In the United Kingdom they are mature from August to November.At that stage the fruiting bodies are yellow to deep brown, and they arerather flat or slightly depressed. As stated above, the fruiting bodiesobtainable from the publiclyavailable specimens ACC 3659 (FPRL 6H) andACC 3253 (Armillaria mellea from Centraabureau voor Schimmelcultures,Baarn, Netherlands) are sources of AM protease.

The mature fruiting bodies may be homogenized with water in a mechanicalblender at 2C. to 4C. The resulting mixture is then separated byfiltration or centrifugation, and both phases (i.e. solid phase andaqueous phase) are retained. The majority of AM protease transfers tothe aqueous phase at this stage. Optionally, the solid phase may beeither (a) freeze-dried, ground with solid carbon dioxide and thenextracted with water at 2C. to 4C., or (b) the solid phase may be groundtogether with a suitable abrasive material, (e.g. sand, and water at 2C.to 4C. and the resulting mixture separated by filtration orcentrifugation. In either case, some AM protease is generally obtainedin the second aqueous extract. The aqueous extract, or the combinedaqueous extracts, as the case may be, is or are then protease byfreeze-dried for storage or it or they can be used directly in stage(ii). A certain amount of darkening of the aqueous extract may occur.This is probably due to the action of a polyphenol oxidase. Thisdarkening can 5 be inhibited by working under nitrogen whereverpracticable and by including in the extraction water at least onetyrosinase inhibitor, for example 10' to 10' M sodium benzoate or 10' to10 M sodium ascorbate.

ii. Water-miscible organic solvents which can be used for precipitatingthe unwanted proteins are, for example, alkanols of up to three carbonatoms or alkanones of up to four carbon atoms, for example ethanol,n-propanol, isopropanol or acetone, or mixtures of any of these. Theseorganic solvents may optionally be used together with a dialkyl ether ofup to five carbon atoms, for example diethyl ether. The precipitation isgenerally carried out at 10C. to 10C., and more particularly at 2C. to2C., and at a pH of 5 to 7, depending on the solvent used. An excessprecipitated is collected by any conventional method,

for example filtration or centrifugation, whereafter it is optionallybut preferably washed with a suitable solvent, for example ice-coldpercent v/v aqueous ethanol. The crude AM protease may optionally thenbe (a) dissolved in ice-cold distilled water and freezedried, or (b)successively washed at 2C. to 2C. with dry ethanol and diethyl ether andthen dried in vacuo at 2C. to 2C.

iv. The crude AM protease so obtained still contains relatively largeamounts of unwanted proteins, and some of these can be removed byexploiting the different affinities of AM protease and the unwantedproteins for certain cation-exchange chromatographic materials, forexample materials containing carboxymethyl radicals attached to apolysaccharide matrix, for example carboxymethylcellulose. The column iseluted with an aqueous buffer of pH 5 to 7, for example pH 6, and at atemperature of about 2C. Generally speaking, the impure AM protease soobtained (still containing some unwanted proteins) is eluted after theprincipal peak of unwanted proteins. The eluted fractions containingimpure AM protease may be concentrated by, for example, pressuredialysis.

v. The remaining unwanted proteinaceous contaminants are removed fromthe impure AM protease by means of a molecular sieve technique at about2C. using a column or membrane of a cross-linked hydrophilic polymerhaving molecular sieve characteristics in respect of a molecular weightof about 30,000. Suitable polymeric materials are, for example,cross-linked dextrans, for example Sephadex (Trade Mark) G75,polyacrylamide gels, for example Bio-gel (Trade Mark), and agaroses, forexample Sagarose (Trade Mark) or Sepharose (Trade Mark). The impure AMprotease from step (iv) is dissolved in a small volume of a buffer of pH6 to 8, for example pH 7, for example a pH 7 0.3M sodiumchloride-phosphate buffer, at about 2C., and the solution is applied tothe column or membrane at about 2C. The column is then eluted with thesame or alike buffer at about 2C., whereupon AM protease is elutedbefore the contaminating proteins.

The eluate containing AM protease may be concentrated by pressuredialysis. in the case of a membrane the contaminating proteins, solventand electrolyte pass through the membrane whereas the AMprotease doesnot; it is preferable to prevent the AM protease from drying outcompletely, otherwise it is difficult to remove from the membrane. Thesolution of AM protease on the application side of the membrane may beconcentrated by pressure dialysis.

According to a further feature of the invention there is provided aprocess for the manufacture of AM protease which comprises the followingsequence of steps:

i. homogenizing mature fruiting bodies of Armillaria mellea with waterin a mechanical blender at 2C. to 4C., separating the resulting mixtureby filtration or centrifugation so as to obtain a solid phase and anaqueous phase (A); an optionally either (a) freeze-drying the said solidphase, grinding the freeze-dried material withsolid carbon dioxide, andextracting the resulting material with water at 2C. to 4C., so as toobtain an aqueous phase (B); or (b) grinding the said solid phase withsand, or a like abrasive material, and water at 2C. to 4C., andseparating the resulting mixture by filtration or centrifugation, so asto obtain an aqueous phase (C); and optionally wherever practicable theoperations under (i) being carried out under nitrogen and the extractionwater containing at least one tyrosinase inhibitor, for example l0" to Msodium benzoate or 10 to 10- M sodium ascorbate;

ii. precipitating unwanted contaminating proteins from the said aqueousphase (A), (B) or (C) by adding thereto at least one organic solventselected from alkanols of up to three carbon atoms and alkanones of upto four carbon atoms, and mixtures thereof, optionally together with adialkyl ether of up to five carbon atoms,

at -2C. to 2C. and at a pH of 5 to 7; and then removingthe precipitatedunwanted proteins by filtration or centrifugation; so as to obtain avsolution comprising AM protease;

iii. precipitating crude AM proteaseby adding to the said solutioncomprising AM protease at least one organic solvent selected fromalkanols of up to three carbon atoms and alkanones of up to four carbonatoms,

- and mixtures thereof, optionally together with a dialkyl ether of upto five carbon atoms,.at -2C. to 2C. and at a pH of 5 to 7; andcollectingthe precipitated crude AM protease by filtration orcentrifugation; and optionally but preferably washing the said crude AMprotease with ice-cold 70 percent v'/v aqueous ethanol;

eluted with an aqueous buffer of pH 5 to 7, for example pH 6, at about2C.; and concentrating the eluted fractions containing AM protease bypressure dialysis; and

v. purifying the impure AM protease so produced by dissolving it in asmall volume of a buffer of pH 6 to 8, for example pH 7, at about 2C.,and applying the resulting solution at about 2C. to a column or membraneof a cross-linked hydrophilic polymer having molecular sievecharacteristics in respect of a molecular weight of about 30,000; andthen either eluting the column with the same or a like buffer at about2C., and concentrating the eluate containing AM protease by pressuredialysis, or, in the case of the said membrane, causing thecontaminating proteins and most of the solvent and electrolyte to passthrough the membrane, and recovering the resulting solution of AMprotease from the application side of the membrane and optionallyconcentrating this solution by pressure dialysis.

According to a further feature of the invention there is provided aprocess for the manufacture of AM protease which comprises growingmycelium of Armillaria mellea on a nutrient medium at about 25C. andthen isolating AM protease from the resulting mycelium by a process asdescribed above except that the said I mature fruiting bodies arereplaced by the said resultand then optionally either (a) dissolving thecrude AM protease in ice-cold distilled water and freeze-drying thesolution, or (b) washing the crude AM protease at -2C. to 2C.successively with dry ethanol and diethyl ether and drying the productin vacuo at 2C. to 2C.;

iv. partially purifying the crude AM protease by chromatography thereofon a cation-exchange chr0- matographic material which containscarboxymethyl radicals attached to a polysaccharide matrix, for examplecarboxymethyl-cellulose; the said material being ing mycelium.

The mycelium may be grown by surface culture at about 25C. on a nutrientmedium comprising maize and wort. As suitable samples of Armillariamellea there may be mentioned, for example, the said samples having ourreference ACC 3253 and 3659.

' AM protease is preferably stored in aqueous solution at about 25C.

According to a further feature of the invention there are providedpharmaceutical compositions comprising AM protease and an inert,non-toxic, pharmaceutically-acceptable diluent or carrier.

The said pharmaceutical compositions may be in a form suitable forsystemic intravenous or intra-arterial infusion or local intravenous orintra-arterial injection. Suitable compositions are sterile injectableaqueous solutions containing 0.05mg. to 1.0mg. of AM protease per ml.,for example 0.25mg. of AM protease per ml. Thepharmaceuticalcompositions of the invention contain conventionaldiluents or carriers and they are made by conventional methods. Theyimayoptionally contain a stabilizing agent, for example human plasmaalbumin, and they may also optionally contain a known analgesic agent.

The pharmaceutical compositions of the invention may be used for:

l. the treatment of arterial thrombosis;

2. the treatment of arterial emboli, for example emboli in the renal ormesenteric arteries, and particularly the leg arteries, and in the aortaat its bifurcation;

3. the treatment of deep vein thrombosis; and

4. the treatment of pulmonary embolism.

It is convenient todiscuss the mode of use of AM protease in terms of(a) systemic therapy, and (b) local intravascular perfusion.

a. Systemic Therapy With AM Protease The resistance of human plasma toAM protease varies between individuals as it does between animals ofdifferent species. This resistance also varies in individual humanpatients during systemic treatment with AM protease. A loading dose isrequired to overcome most of the circulating inhibitor. This close isdetermined in the main by prior titration of the patients plasma invitro and also by titration in vivo by observing the response ofincreasing fractions of the predicted dose of AM protease as it isinfused intravenously. The response which is measured is theprolongation of the clotting time which is produced by AM protease whichis in excess of that affected by the inhibitor. In most cases theloading dose will be 0.05 to mg./kg./hour of AM protease.

When optimum plasma activity of AM protease has been produced, this hasto be maintained by intermittent intravenous dosing of AM protease. Thisis necessary because it appears that there is a return of inhibitor intothe circulation which causes the clotting times to return towards normalvalues, indicating loss of enzymic activity. We have observed thatthrombolytic activity is maintained by administering approximatelypercent of the initial dose of AM protease hourly for the next 2 hours,and then decreasing to less than 1 per- I cent of the initial dose of AMprotease every 2 to 3 hours. The reduction in the rate of dosingrequired to maintain activity is probably due to a fall-off in the rateof return of inhibitor into the circulation.

The duration of treatment with AM protease depends upon the age of thethrombus or thrombi, and their size. Systemic treatment is continueduntil there is evidence of clinical improvement, i.e. return ofpulsation and improvement in the color of the limb in which thecirculation has been affected by an arterial thrombosis. I b. Locallntravascular Perfusion of AM Protease Local perfusion of AM proteasewould be useful in cases where there is a thrombotic occlusion of alarge peripheral vein, or in thrombosed vessels used in the Scribnershunt for renal dialysis, or in a suitable segment of an artery whichhas been blocked by a thrombus. Such a procedure of local perfusion hasthe advantage that the dose used would be considerably less than thatused for systemic therapy, i.e. one-eightieth to one one-hundredth ofthe systemic dose. Also there is no need to assess the level ofinhibitor in the patient's blood. A standard dose can therefore begiven, for example 5 to 10ml. (containing one-eightieth to oneonehundredth of the systemic dose) can be injected into a catheter inthe part of the vein distal to the occlusion. This can be left for 10minutes before aspirating the contents of the catheter. This procedureis repeated two or three times, and in most cases the thrombus will thenbe lyzed. The same procedure can be used for an accessible artery whichis totally occluded by a thrombus, except that the catheter is insertedproximal to the occluded segment.

AM protease as obtained from Armillaria mellea contains a metal. We havefound that the presence of a metal, but not necessarily the metalpresent in the said substance as obtained from Armillaria mellea, isessential for enzymic activity. The apo enzyme can be obtained bytreating the hole enzyme, i.e. AM protease, with 10' M ethylenediaminetetra-acetic acid (pH 7) for 30 minutes at 4C. Excess ethylenediaminetetraacetic acid is removed by dialysis against 0.05 M pH 7.4 209buffer, which has the following composition:

Tris(hydroxymcthyl)aminomcthnnc 6.05g./l. in water Sufficient Mhydrochloric acid to give pH 7.4

Enzymic activity can be restored by treatment of the apo enzyme in anaqueous medium with Co t, Zn Ni Fe and (in this case the enzyme activityis restored to a lesser extent) Cu the products obtained by the saidtreatment will be referred to hereinafter as modified AM protease.

According to a further feature of the invention there are providedpharmaceutical compositions comprising modified AM protease and aninert, non-toxic, pharmaceutically-acceptable diluent or carrier.

Suitable compositions and modes of administration are those compositionsand modes of administration described above in the context of AMprotease itself.

The invention is illustrated by the following examples:

EXAMPLE 1 Stage (i) lOOg. of mature fruiting bodies of Armillaria melleawere homogenized in a M.S.E. Atomix (Atomix is a Trade Mark) blender at2C. with 150ml. of water. The homogenate was mixed with another 150ml.of water, the mixture was stirred at 2C. for 30 minutes, and thencentrifuged. The centrifugate (liquid phase) was kept at 2C. untilfurther processing was effected (see below). The solid residue wasfreeze-dried, the product was ground to a fine powder together withsolid carbon dioxide, the mixture was extracted with 150ml. of water at2C., and the extract was centrifuged. The centrifugate (liquid phase)was combined with the abovementioned centrifugate. (Comments on thisstage: The aqueous extract could be used directly in the next stage, orit could be freeze-dried for storage. Most of the AM protease, aboutpercent, is extracted on the homogenization with water).

Stage (ii) The aqueous solution was adjusted to pH 6.0 with M aceticacid, and the solution was cooled to 2C. in a refrigerated bath. 450ml.of ethanol at 2C. were slowly added to the aqueous solution. The mixturewas well stirred during this addition but care was taken to avoidfrothing. The temperature was kept at 2C. When the addition was completethe mixture was kept at 2C. for 1 hour, and the precipitate which formedwas removed by centrifugation (2,000 X g) for 30 minutes at 2C. Thesolid residue was discarded and the centrifugate was used in stage(iii).

Stage (iii) The centrifugate from stage (ii) was placed in therefrigerated bath and 450ml. of ethanol were slowly added. Thetemperature was allowed to fall to -5C. during this addition. As instage (ii), the mixture was well stirred but frothing was avoided. Whenthe addition was complete the mixture was stirred for 1 hour at -5C. ltwas then centrifuged and the centrifugate was discarded. The solidresidue was suspended in 75ml. of 70 percent v/v ethanol-water at 5C.,and the suspension was centrifuged. The solid residue was dissolved at2C. in 30ml. of water, the solution was shell frozen at 70C. and thenfreeze-dried. (Comments on this stage: About -95 percent of the startingactivity is recovered in this step, and the freeze-dried materialconstitutes some percent by weight of the solid starting material inthis step, i.e. obtained from the aqueous extract). Stage (iv) 100g. ofWhatman microgranular carboxymethylcellulose (CM 52; Whatman is a TradeMark) were washed with a pH 6.0 0.025M sodium citrate buffer until thewashings had pH 6.0. The buffer was prepared by adjusting a solution of7.35g./l. of trisodium citrate dihydrate in water to pl-1 6.0 by theaddition of 2M hydrochloric acid. The equilibratedcarboxymethylcellulose was then packed into a 2.5 X 30cm. column. 2ml.

of a solution of 200mg. of the freeze-dried product of stage (iii) inthe abovementioned citrate buffer were applied to the column at2C. Thecolumn was eluted at 2C. with the citrate buffer at about 20ml./hr. and5ml. fractions were collected. The eluate was monitored for lightabsorbance at 280mg; this gave an indication of the position of all.protein peaks (proteins exhibit a peak of absorbance at 280m The enzymicactivity, i.e. proteolytic activity, was monitored by the methodoutlined above under (l)(c). A large peak of ultraviolet-absorbingmaterial exhibiting no proteolytic activity preceded theproteolytically-active fractions (i.e. containing AM protease), and thelatter fractions were followed by further ultraviolet-absorbingnonproteolytic material. The pooled fractions were concentrated bypressure dialysis at 2C. using Amicon UM l0 membranes (Amicon is a TradeMark). (Comments on this stage: Depending upon the quality of thecarboxymethylcellulose, which varies somewhat, the protein-aceousmaterial including AM protease can be isolated as a discrete peak withabout-20-fold purification or, under less favorable conditions, a fivetofold purification. Up to 90 percent of the AM protease applied to thecolumn is recovered. It may be necessary to alter the salt concentrationof the eluant to compensate for the variable properties of differentbatches of carboxymethyl-cellulose). Stage (v) 10g. of "Sephadex G75(Sephadex is a Trade Mark) in head form were allowed to 'swell for'24hours in an excess of 0.15M sodium chloride 0.05M sodium phosphatebuffer of pH 7.0. This buffer had the following composition:

8.53 g./l. in water 7.8 g./l. in water The swollen material was packedinto a 1.5 X cm. column, and the column was washed with about 100ml. ofthe phosphate buffer at 2C. for 2 hours. 2ml. of a concentrated sampleofthe eluate from stage (iv) were applied to the column. The columnwaseluted at 29C. with the above phosphate buffer at about 20ml./hr.,and 2m]. fractions werecollected. The proteolytic activity of thefractions was monitored as outlined above in stage (iv), and theactivity was found in the first peak of protein eluted. This wasfollowed by another peak of protein which was not proteolytic. Theactive fractions were combined, dialyzed against water at 2C. and

freeze-dried. The freeze-dried product so obtained was AM protease.(Comments on this stage: The purification achieved varies between twoandfive-fold; in general it is lower when stage (iv) is most efficient).

The overall purification achieved in all five stages was 300-500 fold,and about 70 percent of the original proteolytic activity was recovered.

EXAMPLE 2 A mycelial inoculum of Armillaria mellea ACC 3253 was made asfollows:

2 percent malt agar (45ml.) in a medical flat bottle (200ml.) wasinoculated by means of a sterile loop from a master culture ofArmillaria mellea (kept on malt agar in a test tube). The bottle and itscontents were incubated at C. for 14 days. Sterile distilled water (m1.)was added to the bottle and the aerial mycelium was rubbed off with asterile needle to produce a mycelial suspension.

Cooked flaked maize (200g) was placed in glass jars (2 1.). Unhoppedbrewery wort (specific gravity 1.182) was added in sufficient volume toreach the level of the surface of the maize. The mouth of each jar wascovered with aluminum foil, and then the jars and their contents wereautoclaved at 120C. and a pressure of 15lb per sq. inch. After cooling,more wort was added to bring the wort level with the top of the maize.The

25 jars and'contents were autoclaved at 120C. for 20 minutes.

After cooling to room temperature, the maize wort medium was inoculatedwith the mycelial suspension of Armillaria mellea (5ml.), and thenincubated in the dark at 25C. for 12 weeks, and then in the light for 4weeks. The surface layer of mycelium which had formed was scraped offand ground with solid carbon dioxide to give a powder. Phosphate bufferof pH 7.4 I

(2ml.) was added. The buffer consisted of 0. l M sodium dihydrogenphosphate solution and 0.15M sodium 1 chloride solution, adjusted to pH7.4 by the addition of 2M sodium hydroxide solution. The mixture wascentrifuged and the supernatant liquid was retained. There was thusobtained an aqueous solution containing AM protease. 100p]. of the saidaqueous solution containing AM protease were added to 05ml. of asolution of human fibrinogen (95 percent clottable protein; 3mg./ml. inphosphate saline buffer; 0.1M phosphate pH 7.4), followed by l0p.l. of asolution of bovine thrombin (500 N.l.H.* units/ml.) in the same buffer.The clot which formed lysed in about seconds. This result demonstratedthat the said aqueous solution contained at fibrinolytic enzyme. *TheNational lNstitutes of Health, Bethesda, Maryland, U.S.A.

What we claim is:

1. AM protease obtained from Armillaria mellea which is characterized bythe following properties:

a. AM protease catalyzes the hydrolytic degradation of fibrinogen andfibrin.

b. AM protease behaves as a single component on passage through a columnof cross-linked dextran gel which is permeable to proteins of amolecular weight of up to 400,000, and the volume of 0.3M NaCi 0.05M pH7.4 tris/HCl buffer in which at 2C. AM protease emerges from the columncorresponds to that required to elute a protein with a molecular weightof about 30,000.

c. AM protease behaves as a single component on sedimentation at 26 X 10X g in an analytical ultracentrifuge.

. Upon electrophoresis of AM protease for 1 hour at '2 mA/sq; cm. in apolyacrylamide gel, having an acrylamide to bisacrylamide ratio of150:1, and using a pH 4.3 0.3 M B-alanine acetic acid buffer, only onecomponent is evident and this has a migration corresponding to a'y-globin.

e. The N terminal amino acid of AM protease is isoleucine.

f. AM protease readily degrades casein at pH 7 producing fragmentssoluble in trichloro-acetic acid; the degree of digestion is much lessthan that produced by trypsin, chymotrypsin or plasmin. AM proteasecauses little degradation of serum albumin or 'y-globulin, even onprolonged incubation. AM protease causes extensive degradation offibrinogen. AM protease has no action on the following small molecularweight substrates which are commonly used to characterize trypsin-likeor chymotrypsin-like enzymes: a-N-acetylglycyl-L- lysine methyl ester,a-N-acetyl-L-lysine methyl ester, a-N-p-toluenesulphonylL-argininemethyl ester, a-N-acetyl-L-tyrosine ethyl ester and L-typtophan ethylester. AM protease acts upon the B- chain of oxidized insulin causingthe following amino end groups to appear: tyrosine, lysine and, to alimited extent, leucine.

2. A process for the manufacture of AM protease which comprises thefollowing sequence of steps:

a. homogenizing mature fruiting bodies of Armillaria mellea with waterin a mechanical blender at 2C. to 4C., separating the resulting mixtureby filtration or centrifugation so as to obtain a solid phase and anaqueous phase (A); and optionally either (i) freeze-drying the saidsolid phase, grinding the freeze-dried material with solid carbondioxide, and extracting the resulting material with water at 2C. to 4C.,so as to obtain an aqueous phase (B), or (ii) grinding the said solidphase with sand, or a like abrasive material, and water at 2C. to 4C.,and separating the resulting mixture by filtration or centrifugation, soas to obtain an aqueous phase (C); and optionally wherever practicablecarrying out the operations under (a) in a nitrogen atmosphere and theextraction water containing at least one tyrosinase inhibitor;

b. precipitating unwanted contaminating proteins from the said aqueousphase (A), (B) or (C) by adding thereto at least one organic solventselected from alkanols of up to three carbon atoms and alkanones of upto four carbon atoms and mixtures thereof, optionally together with adialkyl ether of up to five carbon atoms, at 2C. to 2C.

and at a pH of 5 to 7; and then removing the precipitated unwantedproteins by filtration or centrifugation; so as to obtain a solutioncomprising AM protease;

. precipitating crude AM protease by adding to the said solutioncomprising AM protease at least one organic solvent selected fromalkanols of up to three carbon atoms and alkanones of up to four carbonatoms and mixtures thereof, optionally together with a dialkyl ether ofup to five carbon atoms, at 2C. to 2C. and at a pH of 5 to 7; andcollecting the precipitated crude AM protease by filtration orcentrifugation; and washing the said crude AM protease with ice-coldpercent v/v aqueous ethanol; partially purifying the said crude AMprotease by chromatography thereof on a cation-exchange chromatographicmaterial containing carboxymethyl radicals attached to a polysaccharidematrix, the said material being eluted with an aqueous buffer of pH 5 to7 at about 2C; and concentrating the eluted fractions containing AMprotease by pressure dialysis; and

. purifying the impure AM protease so produced by dissolving it in asmall volume of a buffer of pH 6 to 8 at about 2C., and applying theresulting solution at about 2C. to a column or membrane of across-linked hydrophilic polymer having molecular sieve characteristicsin respect of a molecular weight of about 30,000; and then eithereluting the column with the same or a like buffer at about 2C. andconcentrating the eluate containing AM protease by pressure dialysis,or, in the case of the said membrane, causing the contaminating proteinsand most of the solvent and electrolyte to pass through the membrane,and recovering the resulting solution of AM protease from theapplication side of the membrane.

3. A process for the manufacture of AM protease which comprises growingmycelium of Armillaria mellea on a nutrient medium at about 25C. andthen isolating AM protease from the resulting mycelium by a process asclaimed in claim 3 wherein the said mature fruiting bodies are replacedby the said resulting mycelium.

4. In a process for the manufacture of AM protease, the step whichconsists in growing'mycelium of Armillaria mellea on a nutrient mediumcomprising maize and wort at about 25C.

1. AM protease obtained from Armillaria mellea which is characterized bythe following properties: a. AM protease catalyzes the hydrolyticdegradation of fibrinogen and fibrin. b. AM protease behaves as a singlecomponent on passage through a column of cross-linked dextran gel whichis permeable to proteins of a molecular weight of up to 400,000, and thevolume of 0.3M NaCl 0.05M pH 7.4 tris/HCl buffer in which at 2*C. AMprotease Emerges from the column corresponds to that required to elute aprotein with a molecular weight of about 30,000. c. AM protease behavesas a single component on sedimentation at 26 X 104 X g in an analyticalultracentrifuge. d. Upon electrophoresis of AM protease for 1 hour at 2mA/sq. cm. in a polyacrylamide gel, having an acrylamide tobisacrylamide ratio of 150:1, and using a pH 4.3 0.3 M Beta -alanineacetic acid buffer, only one component is evident and this has amigration corresponding to a gamma -globin. e. The N terminal amino acidof AM protease is isoleucine. f. AM protease readily degrades casein atpH 7 producing fragments soluble in trichloro-acetic acid; the degree ofdigestion is much less than that produced by trypsin, chymotrypsin orplasmin. AM protease causes little degradation of serum albumin or gamma-globulin, even on prolonged incubation. AM protease causes extensivedegradation of fibrinogen. AM protease has no action on the followingsmall molecular weight substrates which are commonly used tocharacterize trypsin-like or chymotrypsin-like enzymes: Alpha-N-acetylglycyl-L-lysine methyl ester, Alpha -N-acetyl-L-lysine methylester, Alpha -N-p-toluenesulphonyl-L-arginine methyl ester, Alpha-N-acetyl-L-tyrosine ethyl ester and L-typtophan ethyl ester. AMprotease acts upon the Beta -chain of oxidized insulin causing thefollowing amino end groups to appear: tyrosine, lysine and, to a limitedextent, leucine.
 2. A process for the manufacture of AM protease whichcomprises the following sequence of steps: a. homogenizing maturefruiting bodies of Armillaria mellea with water in a mechanical blenderat 2*C. to 4*C., separating the resulting mixture by filtration orcentrifugation so as to obtain a solid phase and an aqueous phase (A);and optionally either (i) freeze-drying the said solid phase, grindingthe freeze-dried material with solid carbon dioxide, and extracting theresulting material with water at 2*C. to 4*C., so as to obtain anaqueous phase (B), or (ii) grinding the said solid phase with sand, or alike abrasive material, and water at 2*C. to 4*C., and separating theresulting mixture by filtration or centrifugation, so as to obtain anaqueous phase (C); and optionally wherever practicable carrying out theoperations under (a) in a nitrogen atmosphere and the extraction watercontaining at least one tyrosinase inhibitor; b. precipitating unwantedcontaminating proteins from the said aqueous phase (A), (B) or (C) byadding thereto at least one organic solvent selected from alkanols of upto three carbon atoms and alkanones of up to four carbon atoms andmixtures thereof, optionally together with a dialkyl ether of up to fivecarbon atoms, at -2*C. to 2*C. and at a pH of 5 to 7; and then removingthe precipitated unwanted proteins by filtration or centrifugation; soas to obtain a solution comprising AM protease; c. precipitating crudeAM protease by adding to the said solution comprising AM protease atleast one organic solvent selected from alkanols of up to three carbonatoms and alkanones of up to four carbon atoms and mixtures thereof,optionally together with a dialkyl ether of up to five carbon atoms, at-2*C. to 2*C. and at a pH of 5 to 7; and collecting the precipitatedcrude AM protease by filtration or centrifugation; and washing the saidcrude AM protease with ice-cold 70 percent v/v aqueous ethanol; d.partially purifying the said crude AM protease by chromatography thereofon a cation-exchange chromatographic material containing carboxymethylradicals attached to a polysaccharide matrix, the said material beingeluted with an aqueous buffer of pH 5 to 7 at about 2*C; andconcentrating the eluted fractions Containing AM protease by pressuredialysis; and e. purifying the impure AM protease so produced bydissolving it in a small volume of a buffer of pH 6 to 8 at about 2*C.,and applying the resulting solution at about 2*C. to a column ormembrane of a cross-linked hydrophilic polymer having molecular sievecharacteristics in respect of a molecular weight of about 30,000; andthen either eluting the column with the same or a like buffer at about2*C. and concentrating the eluate containing AM protease by pressuredialysis, or, in the case of the said membrane, causing thecontaminating proteins and most of the solvent and electrolyte to passthrough the membrane, and recovering the resulting solution of AMprotease from the application side of the membrane.
 3. A process for themanufacture of AM protease which comprises growing mycelium ofArmillaria mellea on a nutrient medium at about 25*C. and then isolatingAM protease from the resulting mycelium by a process as claimed in claim3 wherein the said mature fruiting bodies are replaced by the saidresulting mycelium.